Assessment of Chromosome Cassette Pattern of MRSA in Correlation to Coagulase Gene

The resistance of methicillin-resistant staphylococcus aureus (MRSA) to antimicrobials drugs is due to expression of the mecA gene. Current study was conducted on 33 MRSA clinical samples (Cefotaxime and Oxacillin positive). All MRSA isolates were examined using Antibiogram, Minimum Inhibitory concentration (MIC) and PCR to clarify the expression of SCCmec genes and to detect the differences on repeats of coagulase gene (Coa). Results showed that all isolates were 100% resistant against Amoxycillin-clavulanate, Ampicillin and Cefotaxime, 45.5% were resistant against Ciprofloxacin and erythromycin. mecA gene is expressed in all examined isolates (100%). The expression of SCCmec genes showed that 11.11% expressed type I, 45.45% contained type II, 45.45% contained type III, 63.63% were type IV and 5.55% were typeV. All examined isolates harbored and expressed coagulase gene repeats. Coagulase repeats were 27.27% with 5 repeats (81pb), and 72.72% with 4 repeats. In conclusion, the virulence of MRSA strains is increased and gave different antibiogram activities from different global regions and the repeats of Coa gene give no detectable differences among MRSA strains.

MOLECULAR CHARACTERIZATION AND ANTIMICROBIAL SUSCEPTIBILITY OF MRSA ISOLATED FROM CHRONIC HEMODIALYSIS OUTPATIENTS AND THEIR CORRELATION TO MRSA COLONIZATION AMONG HEALTHCARE WORKERS

The carriage of methicillin-resistant Staphylococcus aureus (MRSA) among dialysis patients is remarkable not only in terms of the risks of developing infections, but also in playing a principle part in transmission among dialysis unit staff. The aim of this study was to detect the colonization of Methicillin-sensitive Staphylococcus aureus and MRSA carriage. Also, our aim was to determine the relatedness of MRSA isolates and the potential routes of transmission using PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) in Hemodialysis Unit of El Zagazig General Hospital, a tertiary medical center in Sharqia, Egypt. This study was conducted on 150 chronic hemodialysis outpatients and 200 non clinical control samples including environmental and healthcare workers (HCWs). Antibiotic susceptibility by VITEK-2 and disc diffusion, PCR amplification of mecA, pvl and coa genes and RFLP-PCR were conducted during the study period. In this study 3.3% of the patients and 3.2% of HCWs colonized with pvl positive MRSA. Fifty percent of MRSA isolates showed a single band PCR product amplification of 810bp fragment corresponding to coa gene. Ten distinct MRSA RFLP banding patterns designated as H1-H10 were obtained. The majority of strains belonged to RFLP banding pattern H1 (33.33%).The prevalence of MRSA carriage among hemodialysis patients was 14% and 9.7 % among HCWs with similar polymorphism patterns. The presence of one major coa gene type confirmed the occurrence of hospital acquired-associated MRSA.

MOLECULAR TYPING OF METHICILLIN-SUSCEPTIBLE STAPHYLOCOCCUS AUREUS (MSSA), ISOLATED FROM MONKEYS, BASED ON COAGULASE GENE POLYMORPHISM

Staphylococcus aureus is a very dangerous microorganism that causes more than 100 nosological forms of disease in humans and animals, including pneumonia, skin and soft tissue infections, food toxicoinfections, wound abscess, etc. Numerous studies on genotyping Staphylococcus aureus, isolated from humans, food and bovine mastitis have been carried out. The lack of information on the genotyping of these pathogens detected in monkeys living in captivity served as a stimulus to conduct a similar research, since staphylococcal infections in the primates are widespread. The present study is devoted to the study of the polymorphism of a variable region of the coagulase gene and to the typing of Staphylococcus aureus isolates from monkeys of different species kept at Adler monkey farm. 115 Staphylococcus aureus isolates were studied using phenotypic and molecular genetic methods. Genotyping was performed using PCR, real-time PCR and PCR with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). A real-time PCR analysis allowed to classify all Staphylococcus aureus as methicillin-susceptible staphylococci (MSSA). After amplification of a variable region of the coagulase gene, 4 types of amplicons of 600, 700, 800, and 900 bp were generated. This data demonstrates structural differences of this gene in the studied isolates. The coagulase gene of 900 bp prevailed. The use of the Cfo1 endonuclease allowed to identify 23 different restriction profiles of the coa gene, but only three of them predominated. Staphylococcus aureus bacteria with seven types of coagulase gene were found only in the lungs of monkeys that died of pneumonia. The results obtained suggest that these isolates have tropism for lung tissue. Among Staphylococcus aureus isolated from pneumonia cases, isolates with three types of the coa gene prevailed. Staphylococcus aureus of eleven types of coagulase gene can be attributed to the invasive isolates, since they were detected in the tissues of various organs. Staphylococcal infection in monkeys kept at the monkey farm is caused by genotypically heterogeneous population of Staphylococcus aureus.

Detection of coagulase gene in Staphylococcus aureus from several dairy farms in East Java, Indonesia, by polymerase chain reaction

Aim: This study was conducted to study the coagulase (coa) gene-based genetic diversity of Staphylococcus aureus, isolated from different samples of cattle from three different regions in East Java Province, Indonesia. Materials and Methods: A total of 160 raw milk samples collected in East Java Province, Indonesia, were screened for the presence of S. aureus. The presumptive isolates were confirmed by coa test. The confirmed S. aureus isolates were subjected to coa gene polymerase chain reaction. Results: Of 160 different samples, 20 (12.5%) isolates of S. aureus were confirmed by positive coa test. Of 20 S. aureus isolates, 19 (95%) isolates carried coa gene. Six different genotypes of coa gene, i.e., 440 bp, 510 bp, 547 bp, 680 bp, 740 bp, and 820 bp were obtained. One coa genotypes, 510 bp (10 isolates) were observed in polymorphism to be more prevalent than the others, and the genotype was present in at least one isolates from every region. Conclusion: It can be concluded that coa gene is easily epidemiological tool for detection of variation strain from S. aureus.

Detection of Staphylococcus aureus’s Strain Similarity on Surgical Ward Nurses’s Hand and Nose and Post Operative Wound Infection Using Coa Gene Through PCR-RFLP Method

Staphylococcus aureus (S.aureus) remains to be the most important cause of post operative wound infection. Nursescould become reservoirs to transmit S.aureus through contaminated hands transiently, or through colonized nose.Strain polymorphism could be determined by Restriction Fragment Length Polymorphism (RFLP), using coa gene andrestriction endonuclease enzyme Alu1. There were 30 isolates of S.aureus’s infection, and 20 isolates taken from handsand nose of the nurses in charge. From 50 isolate positive S.aureus, PCR results showed single and multiple bandswithin 300 to 900 base pairs (bp) in length, and multiple bands within 200 to 600 bp. Five out of 30 patients (17%)showed no PCR-RFLP similarity with any of the nurses. Ten out of 15 nurses which hands were positive for S.aureus,has PCR-RFLP similarity with some patients. There was only 1 out of 5 nurses which nose was positive for S.aureus,showed PCR-RFLP similarity with some patients. Statistically, the proportion of the similar PCR-RFLP between thosesamples in this study is 0.12 (12%). Conclusion: Nurses had 12 % PCR-RFLP similarity for S.aureus with post operativewound infection.

Coagulase gene polymorphism and antimicrobial susceptibility of Staphylococcus aureus isolated from bovine subclinical mastitis milk in Sidi-Bel-Abbes, Algeria

The study was conducted to identify and characterize Staphylococcus aureus in raw milk derived from subclinical mastitis in Sidi-Bel-Abbes Algeria. In this paper, we explore the possibility of detection of the coagulase gene (coa), which encodes the coagulase enzyme, by PCR analysis in antibiotic-resistant isolates, with the latex agglutination phenotype and free coagulase.Out of 336 samples of raw milk examined with California mastitis test (CMT) posi-tive; a total of 142 samples were bacteriologically positive with 56.34% Staphylococcus isolates, 21 (26.25%) isolates were confirmed as S.aureus. Nineteen (90.48%) isolates of S.aureus showed free coagulase on the tube agglutination test. Two atypical S.aureus strains (9.52%) were defective for the clumping factor and / or protein A , determined with the Staphytect plus test and the tube coagulase test. The isolates of S.aureus were resistant to penicillin and tetracycline with 76.19%. Two isolates (9.52%) of S.aureus re-sistant to meticillin (MRSA) were detected in this study, with a MIC of ≥4 μg / liter and a cefoxitin screen test with a MIC of ≥8 μg / liter, and 13 (61.9%) isolates were with a multiresistance phenotype. The 21 isolates were sub-jected to PCR amplification of the 3' end of the coa gene, 18 (85.71%) were revealed on a 1% agarose gel with a single band between 547 bp and 875 bp. The use of the PCR genotypic test to identify the profile of the coa gene can be used as an appropriate identification criterion for differentiating coagulases from S.aureus and for understanding their epidemiology.

Molecular characterization and detection of enterotoxins, methicillin resistance genes and antimicrobial resistance of Staphylococcus aureus from fish and ground beef

A total of 120 samples including 40 freshwaterfish(Oncorhynchus mykiss), 40 seawater fish (Sparus aurata) and 40 ground beef samples were examined for the presence of Staphylococcus aureus. The isolates were identified using biochemical tests and a PCR for the species-specific fragment (Sa442) and thermonuclease gene (nucA). The presence of staphylococcal enterotoxin genes (sea, seb, sec, sed and see), toxin genes (eta, etb, tsst), methicillin resistance gene (mecA) and some phenotypic virulence factors was also tested. Genotypic characterization of the isolates was analyzed by PCR-RFLP of the coa gene. Overall, 36 (30%) meat samples were contaminated with S. aureus. Of the 36 isolates, 3 (8.3%) were found to be positive for enterotoxin genes. Only 1 isolate (5.9%) from ground beef had the sea gene. In addition, 1 (12.5%) of the freshwater fish and 1 (9.1%) of the seawater fish carried both the sea and sed genes. The presence of seb, sec, see, eta, etb and tsst was not detected among the isolates of S. aureus. The amplified coa gene revealed five different clusters. Seven and six distinct RFLP patterns were obtained with AluI and HaeIII digestion, respectively. All isolates were found to be positive for slime, hemolytic and DNase activity while 41.7% of them were beta-lactamase positive. The presence of methicillin resistance was neither detected by PCR nor the disk diffusion method. A total of 94.4% of the isolates were resistant to at least one antimicrobial while 44.4% of them were resistant to at least two or more antimicrobials.